*Corresponding Author:
Ramakrishnan Padmini
Department of Biochemistry, Vels Institute of Science, Technology and Advanced Studies, Pallavaram, Chennai, Tamil Nadu 600117, India
E-mail: [email protected]
Date of Received 17 February 2021
Date of Revision 21 April 2022
Date of Acceptance 19 October 2022
Indian J Pharm Sci 2022;84(5):1309-1322  

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Abstract

This research aimed to explore the antioxidant potency of leaf extracts of Syringodium isoetifolium seagrass. In this study, seagrass leaves were collected from the local tribe’s source, dried and extracted with water, alcohol, hydro alcohols, acetone and n-hexane. Phytochemical investigation on extracts was performed using chemical and gas chromatography-mass spectrometric techniques. It reveals that the extracts are enriched with pharmacologically active compounds such as phenol, polyphenol, alkaloid, glycoside, saponin, flavonoids, dicarboxylic acids and other constituents. Later, the crude extracts were estimated for antioxidant activity using different in vitro methods such as 2,2-diphenyl-1-picryl-hydrazyl-hydrate, total antioxidant, nitric oxide, superoxide anion and hydroxyl radical scavenging activities. The result demonstrated that the extracts exhibited higher radical scavenging activity as compared to the ascorbic acid. Among the different extracts, the hydroalcoholic extract had shown an excellent antioxidant activity due to the existence of more potent phytochemicals.

Keywords

Syringodium isoetifolium, leaf extracts, phytochemical screening, antioxidant activity

The ecosystem is the structural and functional component of ecology where the living things interact with each other and the environment[1]. There are many ecosystems existing in the galaxy[2]. Marine ecosystems are the main ecosystems and exist in sea waters[3]. In which, seagrasses are a vital part of the marine ecosystem, found in shallow, sheltered, softbottomed marine waters[4]. They were found all along with the coastal areas of India[5,6]. These are belonging to the families of Cymodoceaceae, Zoseraceae, Hydrocharitaceae and Posidoniaceae[7]. Around the earth, 52 species of seagrasses were found so far in which 14 species were identified in the west and east coastal part of India[8]. The biomass of seagrass has been utilized frequently as food and drug by coastal indigenous people[9]. Seagrasses are well documented for the presence of potent diverse secondary metabolites. There is an immediate need to quantitatively survey the traditional knowledge of seagrasses in areas where they are abundant and serve as an important resource to coastal communities. In folk medicine, seagrasses have been employed for many therapeutic purposes such as skin diseases, fever, wounds, stomach problems, muscle pains and as a remedy against different kinds of rays[10]. They also provide different pharmacological activities like antioxidant[11], anti-microbial[12], antiviral[ 13], against stomach problems[14], anti-diabetic[15], wounds[16], tranquillizer[17] and anticancer[18] activities etc. Human pollution has contributed most to seagrass declines around the world. There is a necessity to take some initiation to preserve the seagrass and ensure the existence of seagrass to the poor people in future. For many decades, herbal medicines have been used in developing countries as the primary source of medical treatments. The root of Cymodocea sp. is eaten as food commonly known as sea sugarcane. Some of Cymodocea sp. are used against malaria, cough and also used as tranquilizers for babies. Halophila ovalis was used by the fishing communities of Cuddalore and Nagapattinam districts of Tamil Nadu, South India as medicine to treat various skin diseases, burns and boils. Cymodocea sp. are being used as a tranquilizer for babies, as soothing helps during pregnancy and against cough and malaria. Halophila stipulacea (Forssk.) Asch., Cymodocea serrulata (R. Br.) Asch and Magnus and Halodule pinifolia (Miki) Hartog possessed effective antimicrobial effects against seven human pathogens[19,20]. From the past few decades, there has been an upsurge in the search for antioxidant drugs derived from plants.

In the review of the above observation and continuation of our interest to identify the therapeutic activity of Syringodium isoetifolium (S. isoetifolium), antioxidant activity was evaluated. The result suggested that all the extracts of S. isoetifolium were shown excellent activity like standard drugs and it is an important milestone in the development of a naturally derived antioxidant drug for various oxidative stress-related diseases.

Materials and Methods

Collection of seagrass:

An excellent quality of S. isoetifolium from the family of Cymodoceaceae was collected from Devipattinam, Ramanathapuram District, Tamilnadu state, India. The collected materials of the plants were authenticated by Dr M. U. Sharief, Scientist E and head, Botanical Survey of India, Southern Regional Centre, T. N. A. U Campus, Coimbatore 641003, India. The Voucher specimen number of the S. isoetifolium is BSI/SRC/5/23/2021/ Tech/372.

Preparation of seagrass extracts:

Collected seagrasses were washed thoroughly with sterile seawater to remove the extraneous dirt, dried in a shade, pulverized and prepared extracts with different solvents of increasing polarity such as distilled water, alcohol, hydro alcohol (30:70), acetone and n-hexane. After drying, the seagrass were sliced into small pieces and then mashed until it becomes a fine powder. The powdered seagrass was immersed in 450 ml of cold solvents in a closed container with gentle shaking for 24 h. These seagrass extracts were collected, filtered and the solvent was eliminated at 60° in the rotary evaporator (model: rotavapor R-210 from Buchi)[21,22]. The solid seagrass extracts have persevered in a dark container (fig. 1). The yield was calculated using the following formula

Percentage Yield=(W1×100)/W2

Here W1-Weight of extract after removing the solvent; W2-Dry weight of the sample.

These plant extracts were utilized for the identification of different phytochemicals present in S. isoetifolium.

Quality control of S. isoetifolium:

Determination of ash value: 3 gm of S. isoetifolium powder was incinerated in a Silica crucible over the burner. The charred material was heated in muffle furnace for 6 h at 600°.The ash formed was white and free from carbon. It was cooled and weighed on the ash less filter paper[23].

Powder fineness and sieve size: Place 100 g of the powder being examined upon the appropriate sieve having a close fitting receiving pan and cover. Shake the sieve in a rotary horizontal direction and vertically by tapping on the hard surface for not less than 20 min. Weigh accurately the amount remaining on the sieve and in the receiving pan[24].

Phytochemical analysis:

The collected dried extracts of S. isoetifolium were analyzed for the presence of different phytochemicals by chemical and Gas Chromatography-Mass Spectrometry (GC-MS) techniques. In the chemical method, phytochemicals were identified through qualitative and quantitative ways using appropriate reagents. Whereas, GC-MS compounds were recognized through their mass value and distinctive mass fragmentation patterns[25].

Chemical method:

Chemicals: All chemical substances were purchased with the highest purity (≥98.0 %) and utilized for the identification of different phytochemicals present in the leaf extract of S. isoetifolium. The chemicals such as Ferric chloride (FeCl3), Olive oil, Folin-Ciocalteu reagent, Aluminium chloride (AlCl3), Ammonia, Sulfuric acid (H2SO4), Hydrochloric acid (HCl), Acetic anhydride, Chloroform, Mayer reagent, Ethanol, Ferricyanide, Glacial acetic acid, Sodium hydroxide (NaOH), Ammonium hydroxide (NH4OH), Gallic acid, Sodium carbonate (Na2CO3), Ammonium chloride (NH4Cl), Methanol, Potassium acetate (CH3COOK), Folin-Denis reagent, 2,2-Diphenyl-1-Picryl-Hydrazyl- Hydrate (DPPH), Sodium phosphate, Ammonium molybdate, Sodium nitroprusside, Phosphate buffer, Sulfanilic acid reagent, 1,10-phenanthroline, Butylated Hydroxyl Toluene (BHT) etc., were all purchased from Merck, United States of America (USA).

Qualitative phytochemicals screening:

Chemical tests were performed with different reagents using standard procedures to know the phytochemicals present in the leaf extracts of S. isoetifolium[26].

Quantitative analysis of phytochemicals:

Total Phenolic Content (TPC): Approximately 1 ml of seagrass extract or Gallic acid standard phenolic compound was mixed to 9 ml of distilled water in a volumetric flask. About 1 ml of Folin-Ciocalteu’s phenol reagent was added and blended gently. After a few minutes, nearly 10 ml of 7 % Na2CO3 solution was admixed into the above solution. The solution was diluted to 25 ml of purified water. The concoction was kept aside for 1.5 h at 23°, after which the absorbance was examined at 750 nm. The TPC was calculated from the extrapolation of calibration curve which was made from the different concentrations of seagrass/gallic acid solutions (20 to 80 μg/ml). The TPC was expressed as milligrams of Gallic acid equivalents per gram in a dried seagrass sample[27].

Total Flavonoids Content (TFC): The TFC was conducted according to Hossain et al.[28] using the colorimetric method. About 0.5 ml of seagrass extracts were mixed with 1.5 ml of methyl alcohol, 0.1 ml 10 % AlCl3, 0.1 ml 1 M CH3COOK and 2.8 ml of water. The above mixture remained at 20°-25° for 30 min and absorbance was measured at 510 nm. The calibration curve was made using quercetin solutions at the concentration of 20 to 80 μg/ml in methyl alcohol. TFC was disclosed as mg of QE/g of dry weight[28].

Total Saponins Content (TSC): The TSC in different concentrations of plant extracts were estimated by colorimetric methods. 0.25 ml of seagrass extract admixed with 1 ml of reagent mixture (H2SO4/glacial acetic acid 1:1 v/v). The content was heated at 60° for 30 min in a water bath and kept aside for some time. The absorbance of seagrass extract was calculated at a wavelength of 527 nm using a spectrophotometer. The TSC was expressed as 100 g-1 of oleanolic acid equivalents[29].

Total Tannin Content (TTC): The TTC was performed according to Medini et al.[30] method. 0.2 ml of seagrass extract admixed with 0.5 ml of Folin-Denis reagent, 1 ml of Na2CO3 solution and 7.5 ml of water. The content of the concoction was mixed well, kept aside for 30 min and the absorbance was measured at 760 nm. TTC was expressed as milligrams of tannic acid equivalents per gram of dried sample. The results were evaluated by correlation coefficient (R2) and linear regression and using Microsoft excel[30].

GC-MS analysis:

GC-MS analysis of aqueous, ethanol and hydro alcoholic extracts of S. isoetifolium was done at SRM institute of science and technology, Kattankulathur, Chennai using Agilent 7890B GC connected to 5977A mass selective detector, furnished with Electron Ionization (EI) and a fused phenyl methyl silox column HP-5 (30 m×250 μm×0.25 μm film thickness) was used. The oven temperature was modified from -60°-325° for 55 min. Highly pure helium gas was used as carrier for this study. The flow rate of carrier gas was fixed 1 ml/min, for sample injection of 1 μl and the ionization voltage of MS-analysis was controlled by EI procedure at 70 eV[31].

Antioxidant activity:

Preparation of sample: S. isoetifolium leaves were airdried at room temperature and pulverized into powder for extraction. The powder (20 g) was macerated in 1000 ml of hydro alcohol (30:70 %), shake vigorously for about 1 h using a rotatory shaker. The mixture was kept aside for 24 h and then the extracts were filtered with Whatman filter paper (No.1). The filtrate was drie concentrations (20, 40, 60 and 80 μg/ml) for in vitro antioxidant activity[31].

DPPH radical-scavenging activity:

The activity was done according to Gopi et al.,[32]. Take a 2 ml DPPH methyl alcohol solution (25 μg/ ml) and mix with 0.5 ml seagrass extracts at various concentrations. The above content was stirred and allowed to rest at 30°-35° for 30 min. The absorbance was calculated at 517 nm.

Percentage radical scavenging activity=100-(Ac-As)/ Ac×100

Where AC=Absorbance in control; AS=Absorbance of extracts.

Total Antioxidant Capacity (TAC):

The phosphomolybdate technique has been employed regularly to assess the TAC of seagrass extracts[33]. In the existence of seagrass extracts, a green-colored phosphomolybdenum V complex formed, which shows absorbance at 695 nm. 0.3 ml of seagrass extracts was mixed with 3 ml of reagent (28 M sodium phosphate, 0.6 M H2SO4 and 4 M ammonium molybdate) in a tube. It was incubated at 95° for 1.5 h. The absorbance was measured at 695 nm. Methyl alcohol (0.3 ml) is used as a blank[31]. The TAC was calculated according to the following equation Percentage inhibition=A0-A1/A0×100

Where A0=Absorbance of the control and A1=Absorbance of the extracts.

Superoxide anion scavenging activity:

It was measured according to Pavithra et al.[34]. In this experiment, the superoxide anion was produced in 3 ml of Tris-HCl buffer (100 mM, pH 7.4) containing 0.75 ml of Nicotinamide Adenine Dinucleotide (NADH) (936 μM) solution, 0.75 ml of nitro blue tetrazolium chloride (300 μM) solution and 0.3 ml of various concentrations of the extracts. The reaction started with the addition of 0.75 ml of phenazine methosulfate (120 μM) to the mixture. After 5 min at room temperature, the absorbance was measured at 560 nm. The activity was calculated according to the following formula

Percentage inhibition=A0-A1/A0×100

Where A0=Absorbance of the control and A1=Absorbance of the seagrass extracts.

Nitric Oxide (NO) scavenging activity:

The NO scavenging activity was determined according to Ali et al.,[35]. Sodium nitroprusside in an aqueous solution produces NO, which reacts with oxygen to generate Nitrite ions (NO2-). Take 2 ml of 10 mM sodium nitroprusside in 0.5 ml phosphate buffer saline (pH 7.4) was mixed with 0.5 ml of seagrass extracts at different concentrations and the mixture was incubated at 25° for 2.5 h. After incubation, 0.5 ml was taken out and added into a 1.0 ml sulfanilic acid reagent (33 % of sulfanilic acid in 20 % glacial acetic acid) and incubated at 30°-35° for 5 min. Finally, 1.0 ml naphthyl ethylenediamine dihydrochloride (0.1 % w/v) was added and incubated 30°-35° for 30 min. The absorbance was measured at 540 nm using a spectrophotometer. The scavenging activity was measured by using the following equation

Percentage inhibition=A0-A1/A0×100

Where A0=Absorbance of the control and A1=Absorbance of the extracts.

Hydroxyl radical scavenging activity:

Hydroxyl radicals scavenging activity was calculated by Fenton reaction according to Sowndhararajan et al.[36]. The hydrogen peroxide was added to the reaction mixture containing 90 μl of 1 mM 1, 10-phenanthroline, 60 μl of 1.0 mM FeCl3, 2.4 ml of 0.2 M phosphate buffer (pH 7.8), 150 μl of 0.17 M H2O2 and 1.5 ml of seagrass extracts at different concentrations. After incubation at 30°-35° for 5 min, the absorbance was measured at 560 nm using a spectrophotometer. The radical (OH.) scavenging activity was calculated using the following formula

Percentage inhibition=A0-A1/A0×100

Where A0=Absorbance of the control and A1=Absorbance of the extracts.

Statistical method:

All the scavenging activities were done in triplicates. The quantity of seagrass required to inhibit radicals concentration by 50 % was graphically calculated by a linear regression method using GraphPad Instat software (version 3). The result is shown as graphically/mean±standard deviation.

Results and Discussion

An excellent quality of leaf of S. isoetifolium from the family of Cymodoceaceae was collected from Devipattinam, Ramanathapuram District, Tamilnadu state, India. Collected seagrasses were washed thoroughly with sterile seawater to remove the extraneous dirt, dried in a shade, pulverized into fine powder. It was used to study the quality control test such as ash value and degree of coarseness or fineness of the powder. The ash content of the seagrass was determined using a dry ashing method. It shows leaf of S. isoetifolium contained ash value of 29.42 %±0.35 %. The degree of coarseness or fineness of a powder is differentiated by nominal aperture size of the mesh of the sieve through which the powder is able to pass, expressed in μm. The particles of the powder which pass through a No. 355 sieve and not more than 40 % through a No.180 sieve. Therefore, the coarseness or fineness of the leaf powder is moderately fine. The results of this study are in line with the comparative proximate composition values for different species of the seagrasses. The presence of phytochemicals was identified through the chemical methods and GCMS technique. The phytochemicals were confirmed in the chemical method through the qualitative and quantitative tests using different reagents (Table 1). Several phytochemicals such as tannin, saponin, steroids, terpenoids, triterpenoids, anthraquinone, flavonoids, polyphenol, glycosides, alkaloids and coumarins were present in Aqueous Extracts of S. isoetifolium (AESI), Ethanol Extracts of S. isoetifolium (EESI) and Hydro Alcohol Extracts of S. isoetifolium (HAESI), Acetone Extracts of S. isoetifolium (ACESI) and n-Hexane Extracts of S. isoetifolium (HESI) (Table 2). The phytochemical analysis of the S. isoetifolium was examined and summarized in Table 1-Table 8 and fig. 1-fig. 7. This was highly supported by the previously published studies on seagrass[37,38]. Among the different extracts, the hydroalcoholic extract had shown the highest yield of phytochemicals such as TPC (192.44±13.47 mg GAE/g), TFC (106.54±7.45 mg quercetin/g), TSC (52.61±3.68 mg Quillaja/g) and TTC (80.65±5.64 mg tannic acid/g). Moreover, the quantitative analysis data for S. isoetifolium in the present study was found to be consistent with those reported in the past[39-42].

Name of Sample Total phenol (Milligrams GAE equivalents per gram) Flavonoids (Milligrams of quercetin equivalents per gram) Saponin (Milligrams of Quillaja saponin equivalents per gram) Tannin (Milligrams of tannic acid equivalents per gram)
S. isoetifolium 192.44±13.47 106.54±7.45 52.61±3.68 80.65±5.64

Table 1: Quantitative Analysis Of Phenol, Flavonoids, Saponin And Tannin Content Present In The Leaf Extracts Of S. isoetifolium

S. No Phytochemicals Aqueous extract Ethanol extract Hydro-alcoholic extract (30:70 %) Acetone Chloroform
1 Tannin ++ ++ ++ - +
2 Saponin ++ ++ ++ - -
3 Flavonoids + ++ ++ - +
4 Steroids + ++ + - -
5 Terpenoids + + + + -
6 Triterpenoids + + + + -
7 Alkaloids + ++ + - +
8 Anthroquinone + + + - -
9 Polyphenol ++ ++ ++ - -
10 Glycoside + + + + -
11 Coumarins + + + + -
12 Emodins - - - - -
13 Anthocyanins - - - - -
14 Carbohydrate + + + + +
15 Carboxylic acid + + + + +

Table 2: Qualitative Phytochemical Analysis of Leaf Extracts of S. isoetifolium

Peak R. Time Area % Height % MW MF Name of the compounds
AESI-A
1 4.226 89.303 52862499 92 C7H8 Toluene
2 28.607 0.029 99898 296 C21H44 Heptadecane, 2,6,10,,15-teetramethyl phthalic acid
3 32.255 0.033 116198 306 C18H26O4 Hex-3-yl isobutyl ester
4 33.05 0.016 83883 324 C23H48 Heptadecane, 9-hexyl-
5 33.243 0.045 134057 270 C17H34O2 hexadeconic acid methyl ester
6 34.164 0.033 85822 278 C16H22O4 Dibytylphthalate
7 34.58 0.157 141928 256 C16H32O2 n-Hexadeconic acid
8 36.482 0.07 355706 294 C19H34O2 11,14-Octadecadienoic acid methyl ester
9 36.578 0.225 873038 296 C19H36O2 10-Octadecenoic acid methyl ester
10 37.039 0.026 112149 298 C19H38O2 Heptadecanoic acid, 16-methyl-, methyl ester.
11 38.183 0.278 118728 356 C21H40O4 9-Octa decenoic acid (z)-2-hydroxy-1-(hydroxy methyl) ethyl ester
12 40.716 0.074 93685 456 C27H52O5 Dodecanoic acid, 1-hydroxymethyl)-1,-2 ethanediyl ester
13 44.119 0.241 844372 390 C24H38O4 Phthalic acid, di (2-propyl pentyl) ester
14 44.527 0.172 277878 456 C27H52O5 Dodecanoic acid, 1-(hydroxy methyl)-1,2-ethanediyl ester.
EESI-B
1 4.137 56.942 44162888 92 C7H8 Toluene
2 5.021 0.719 876023 269 C16H28ClN Benzyltri-n-propyl ammnonium chloride
3 5.482 0.483 732237 129 C7H15NO N,N dimethyl pivalamide
4 26.104 0.448 787201 200 C12H24O2 Dodecanoic acid
5 30.398 0.506 1002310 228 C14H28O2 Tetra decanoic acid
6 31.111 0.192 385376 196 C12H20O2 9,10-dimethyl tricyclo {4.2.1.1.(2,5)}decane-9-10-diol.
7 31.386 0.041 350738 338 C22H42O2 Phytol, acetate
8 31.579 0.058 381084 268 C18H36O 2-pentadecanone, 6,10,14-trimethyl
9 32.195 0.082 238835 278 C16H22O4 Dibutyl phthalate
10 33.02 0.011 77071 366 C26H54 Octa decane, 3-ethyl-5-(2-ethyl butyl)-
11 33.117 0.046 329843 256 C17H36O 1-Hexadecanol,2-mehtyl-
12 33.183 0.066 490890 270 C17H34O2 Hexadecanoic acid, methyl ester
13 34.067 0.79 1738912 254 C16H30O2 Palmtoleic acid
14 34.543 9.237 18383784 256 C16H32O2 n-Hexadecanoic acid
15 36.036 0.082 143713 268 C17H32O2 Cis-10-heptadecenoic acid
16 36.289 0.065 237470 270 C17H34O2 Heptadecanoic acid
17 36.43 0.068 505440 294 C19H34O2 Methyl 9-cis, 11-trans-octa decadienoate
18 36.534 0.221 1492702 296 C19H36O2 Trand 13-octa decenoic acid, methyl ester
19 36.831 0.189 1026914 296 C20H40O Phytol
20 36.994 0.045 220832 298 C19H38O2 Heptadecanoic acid, 16-methyl ester
21 37.871 19.654 27064423 282 C18H34O2 Cis-vaccenic acid
22 38.168 2.877 13812305 284 C18H36O2 Octadecanoic acid
23 38.502 0.64 1200659 280 C18H32O2 9,12,Octadecadienoic acid
24 38.914 0.063 1491659 310 C20H38O2 Ethanol z-(9,12-octa decadienyloxy)-(z,z)
25 39.193 0.711 1895472 280 C18H32O2 9,12-octadeca dienoic acid (z,z)-
26 41.028 0.996 2585848 310 C20H38O2 Cis-11-eicosenoic acid
27 41.4 0.419` 1535543 312 C20H40O2 Eicosenoic acid
28 42.224 0.024 147908 356 C21H40O4 9-octa decenoic acid, (z)-,2-hydroxy-1-(hydroxymethyl) ethyl ester
29 43.829 0.138 379780 330 C19H38O4 Hexadecanoic acid, 2-hydroxy-1-(hydroxymethyl) ethyl ester.
30 44.081 0.35 2336750 390 C24H38O4 Phthalic acid, di(2-propyl pentyl) ester
31 44.26 2.605 8047321 338 C22H42O2 Erucic acid
32 44.564 0.387 1530826 340 C22H44O2 Docosanoic acid
33 46.555 0.74 1494775 356 C21H40O4 9-octa decenoic acid (z)-,2-hydroxy-1-(hydroxy methyl ethyl ester)
34 47.662 0.086 229287 368 C24H48O4 Tetra cosanoic acid
HAESI-C
1 4.152 35.927 20959817 92 C7H8 Toluene
2 32.3 0.044 86703 320 C19H28O4 Phthalic acid, 5-methyl hex-2-yl isobutyl ester
3 33.273 0.013 57102 270 C17H34O2 Hexadecanoic acid, methyl ester
4 34.179 0.049 103427 376 C23H36O4 Phthalic acid, butyl undecyl ester
5 34.432 0.764 1043999 256 C16H32O2 N-hexadecanoic acid
6 36.519 0.026 132412 294 C19H34O2 Methyl 10-trans, 12-cis-octadecadienoate.
7 36.608 0.073 335007 296 C19H36O2 Trans-13-octa decenoic acid, methyl ester
8 36.898 0.019 83247 296 C20H40O Phytol
9 37.069 0.023 77335 298 C19H38O2 Hepta decanoic acid, 16-methyl-,methyl ester
10 37.782 0.156 218002 282 C18H34O2 Cis-vaccenic acid
11 38.116 0.139 210895 284 C18H36O2 Octa decanoic acid
12 44.148 0.116 338489 390 C24H38O4 Diisooctyl phthalate
13 49.646 43.343 3289601 456 C27H52O5 Dodecanoic acid, 1-(hydroxy methyl)-1,2-ethane diyl ester
14 54.719 2.134 446409 436 C26H44O5 Ethyl iso-allocholate

Table 3: phytochemical screening leaf extracts of S. isoetifolium using GC-MS techniques

Samples (percentage inhibition) Concentrations (µg/ml) IC50 value (µg/ml)
20 µg/ml 40 µg/ml 60 µg/ml 80 µg/ml
S. isoetifolium 23.64±1.65 44.55±3.11 62.73±4.39 79.10±5.53 47.28
Ascorbic acid (Std) 25.46±1.78 47.28±3.30 78.64±5.50 89.55±6.26 40.84

Table 4: DPPH Radical Scavenging Activity of Leaf Extracts of S. isoetifolium and Ascorbic Acid at Different Concentrations

Samples (Percentage inhibition) Concentrations (µg/ml) IC50 value (µg/ml)
20 µg/ml 40 µg/ml 60 µg/ml 80 µg/ml
S. isoetifolium 22.50±1.57 40.31±2.82 65.62±4.59 81.56±5.70 47.53
Ascorbic acid (Std) 26.56±1.85 42.50±2.97 69.37±4.85 84.68±5.92 44.25

Table 5: Total Antioxidant Activity of Leaf Extracts of S. isoetifolium and Ascorbic acid at different Concentrations

Samples (Percentage inhibition) Concentrations (µg/ml) IC50 value (µg/ml)
20 µg/ml 40 µg/ml 60 µg/ml 80 µg/ml
S. isoetifolium 20.35±1.42 45.71±3.19 71.42±4.99 89.28±6.24 44.24
Ascorbic acid (Std) 28.57±1.99 54.64±3.82 75.71±5.29 92.14±6.44 37.94

Table 6: Superoxide anion Radical Scavenging activity of S. isoetifolium and Ascorbic acid at different concentrations

Samples (Percentage inhibition) Concentrations (µg/ml) IC50 value (µg/ml)
20 µg/ml 40 µg/ml 60 µg/ml 80 µg/ml
S. isoetifolium 20.47±1.43 47.14±3.29 69.04±4.83 84.28±5.89 45.09
Ascorbic acid (Std) 23.33±1.63 51.90±3.63 70.47±4.93 91.42±6.39 41.67

Table 7: Nitric Oxide Scavenging Activity of S. isoetifolium and Ascorbic Acid at Different Concentrations

Samples (Percentage inhibition) Concentrations (µg/ml) IC50 value (µg/ml)
20 µg/ml 40 µg/ml 60 µg/ml 80 µg/ml
S. isoetifolium 21.66±1.51 43.33±3.03 68.75±4.81 81.25±5.68 46.32
Ascorbic acid (Std) 25.41±1.77 53.75±3.76 72.91±5.10 93.33±6.53 39.81

Table 8: Hydroxyl Radical Scavenging Activity of S. isoetifolium and Ascorbic Acid at Different Concentrations

isoetifolium

Fig. 1: Image of S. isoetifolium in shallow and (A): Deep water; (B): Shade dry; (C): Liquid extracts and (D): Dried extracts

Phytochemical

Fig. 2: Phytochemical constituents identified in the aqueous, ethanol and hydro alcohol leaf extracts of S. isoetifolium using gas chromatography-mass spectrometry

DPPH

Fig. 3: DPPH radical scavenging activity of different concentrations of leaf extracts of S. isoetifolium Note: Percentage of inhibition of hydro alcoholic extract image and Percentage of inhibition of Ascorbic acid imageat p?0.05

antioxidant

Fig. 4: Total antioxidant activity of different concentrations of hydroalcoholic extracts of S. isoetifolium Note: Percentage of inhibition of sample image and Percentage of inhibition of Ascorbic acid image at p?0.05

Superoxide

Fig. 5: Superoxide anion radical scavenging activity of different concentrations of hydroalcoholic extracts of S. isoetifolium Note: Percentage of inhibition of hydroalcoholic extract image and Percentage of inhibition of Ascorbic acid image at p?0.05

Nitric

Fig. 6: Nitric oxide scavenging activity of different concentration of hydro alcoholic extracts of S. isoetifolium Note: Percentage of inhibition of hydro alcoholic extract image and Percentage of inhibition of Ascorbic acid image at p?0.05

Hydroxyl

Fig. 7: Hydroxyl radical scavenging activity of different concentrations of hydro alcoholic extracts of S. isoetifolium Percentage of inhibition of hydro alcoholic extract image; Percentage of inhibition of Ascorbic acid image at p?0.05

In GC-MS, the existence of different phytochemicals was identified through their Molecular Weight (MW) and Molecular Formula (MF) etc. There were 63 compounds identified in this method from the HAESI extracts (fig. 2 and Table 3). The prevailing compounds found in the mentioned extracts are Hexadecanoic acid methyl ester, Hexadecanoic acid, Ethyl iso-allocholate etc., Phytochemicals present in the plant extract significantly contributes to biological activities. Therefore, it is necessary to identify the different phytochemicals of the plant extract. Earlier reports show that seagrasses have antioxidant activity due to the presence of polyphenols, flavonoids and other phytochemicals[43-45]. Flavonoids are naturally occurring biological compounds that can act as potent antioxidants and can prevent cardiovascular disease by preventing the oxidation of Low-Density Lipoprotein (LDL)[46]. In the review of the above observation and continuation of our interest to identify the therapeutic activity of S. isoetifolium, antioxidant activity was evaluated. Antioxidant activity is a complex procedure and is influenced by different mechanisms, which cannot be identified through a single method. Therefore, it is needed to perform different ways to measure antioxidant activity. Different aliquots of hydroalcoholic extract of S. isoetifolium and ascorbic acid were prepared for in vitro antioxidant activity d for in vitro antioxidant activity such as DPPH, TAC, Superoxide radical scavenging activity, NO scavenging activity and Hydroxyl radical scavenging activities. Minimum Inhibitory Concentration (MIC) is also estimated by calculating the IC50 value. The DPPH radical scavenging activities increased with the increasing concentration (20, 40, 60 and 80 μg/ml) of HAESI extract. The radical scavenging activities of 23.64±1.65, 44.55±3.11, 62.73±4.39, 79.10±5.53 μg/ml respectively at various concentrations (20, 40, 60 and 80 μg/ml) was observed and IC50 was found to be 47.28 μg/ml (Table 4 and fig. 3). IC50 values of the scavenging assay were calculated by plotting the percentage of inhibition against the concentration HAESI extracts. Similarly, the radical scavenging activities of ascorbic acid were performed and the results were between 25.46±1.78 to 89.55±6.26 μg/ ml with increasing concentrations. The IC50 value of the standard drug was found to be 40.84 μg/ml. The DPPH radical scavenging activities of HAESI extracts are competitive when compared to the standard at the given concentrations.

The hydroalcoholic extract showed the significant DPPH scavenging activity with the lowest IC50 value due to the presence of polyphenols compounds (Table 4). DPPH free radical scavenging activity reported in our study of S. isoetifolium is much higher than result reported from other seagrasses[47,48]. The TAC was increased with the increasing concentration (20, 40, 60 and 80 μg/ ml) of HAESI extracts. The total antioxidant activities 22.50±1.57, 40.31±2.82, 65.62±4.59, 81.56±5.70 μg/ml respectively at different concentrations were observed and IC50 was found to be 47.53 μg/ml (Table 5 and fig. 4). Similarly, the total antioxidant radical scavenging activities of ascorbic acid were performed and the result was between 26.56±1.85 to 84.68±5.92 μg/ml with increasing concentration. The IC50 value of the standard drug was found to be 44.25 μg/ml. Superoxide radical is believed to be a chief source of reactive oxygen species in living things. The superoxide radical scavenging activity of different concentrations (20, 40 60 and 80 μg/ml) of HAESI extracts was evaluated with the same dose of ascorbic acid. The HAESI extract showed superoxide anion radical scavenging activities were 20.35±1.42, 45.71±3.19, 71.42±4.99, 89.28±6.24 μg/ml respectively at various concentrations and IC50 was found to be 44.24 μg/ml (Table 6 and fig. 5). In the same way, the superoxide radical scavenging activities of ascorbic acid was performed with the ascorbic acid and the result was between 28.57±1.99 to 92.14±6.44 with increasing concentrations. The IC50 value of the standard drug was found to be 37.94 μg/ml. In another method, NO scavenging activity of HAESI extracts was evaluated with the same doses of ascorbic acid ranging from 20-80 μg/ml. The HAESI extracts showed NO radical scavenging activities 20.47±1.43, 47.14±3.29, 69.04±4.83, 84.28±5.89 μg/ml respectively at different concentrations and IC50 was found to be 45.09 μg/ml (Table 7 and fig. 6). Similarly, the radical NO scavenging activity of ascorbic acid was performed and the result was between 23.33±1.63 to 91.42±6.39 with increasing concentrations. The IC50 value of the standard drug was found to be 41.67 μg/ml.

Athiperumalsami et al.[49] have reported highest antioxidant activity in the methanolic extract of Halophila ovalis than Halodule pinifolia tested by the NO scavenging method. At last, hydroxyl radical scavenging potential of HAESI extracts to inhibit hydroxylradicals (OH) was assessed at various concentrations 20-80 μg/ml. The HAESI extracts showed hydroxyl radical scavenging activities 21.66±1.51, 43.33±3.03, 68.75±4.81, 81.25±5.68 μg/ml respectively at different concentrations and IC50 was found to be 46.32 μg/ml (Table 8 and fig. 7). In the same way, the hydroxyl radical scavenging activity of ascorbic acid was performed and the result was between 25.41±1.77 to 93.33±6.53 μg/ml with increasing concentrations. The IC50 value of the standard drug was found to be 39.81 μg/ml. In agreement with previous studies there was a significant correlation between our antioxidant value and the values in the study of Duan et al.[50]. The result demonstrated that the hydroalcoholic extracts exhibited scavenging and/or reducing effects on DPPH, phosphomolybdate, superoxide, NO, hydroxyl radicals present in the reagents. The radical scavenging activity of hydroalcoholic extract of S. isoetifolium is due to the presence of polyphenols, flavonoids and other phytochemicals. At all concentrations of hydroalcoholic extract from 20 to 80 μg/ml, displayed an excellent antioxidant effect. However, the lowest radical scavenging activity was observed in the lower concentrations of hydroalcoholic extracts. Whereas the high concentration of hydroalcoholic extract exhibits excellent antioxidant activity as similar to that of standard drug. The order of radical scavenging activity of hydroalcoholic extract of S. isoetifolium was found to be 80 μg/ml?60 μg/ml?40 μg/ml?20 μg/ml. Similar results were also obtained in studies conducted by other scientists who were involved in the determination of antioxidant activity in the seagrass at various concentrations[51-53]. All the extracts were less effective than the standard ascorbic acid radical scavenging activity and IC50 values. This present finding corroborate well with earlier reports in other species of seagrass.

In conclusion, the seagrass leaf extracts of S. isoetifolium were screened for phytochemicals by using chemical and GC-MS techniques. It is observed that the presence of phenol, polyphenol, alkaloid, glycoside, saponin, flavonoids, dicarboxylic acids and other constituents in the qualitative analysis. Whereas, quantitative determination shows the presence of TPC (192.44±13.47 mg GAE/g), TFC (106.54±7.45 mg quercetin/g), TSC (52.61±3.68 mg Quillaja/g) and TTC (80.65±5.64 mg tannic acid/g). The GC-MS analysis of the seagrass extract of hydro alcohol revealed the presence of 62 compounds that could contribute to the medicinal property of the plant leaves. Here, the compounds were used to identify the different phytochemicals present in the hydroalcoholic extract from the MF and MW of compounds. In addition to that, the HAESI extracts of S. isoetifolium proved to be the most effective radical scavenger activity in the DPPH, total antioxidant, NO, superoxide anion, hydroxyl radicals etc., the higher radical scavenging activity of HAESI extracts due to the presence of phenol, polyphenols and other compounds. They react with the various species of radicals such as hydroxyl, peroxide, superoxide and NO etc., and reduce their existence. Therefore, extracts of S. isoetifolium can be recommended for various oxidative stress-related diseases. The above result encourages us to isolate and confirm the specific phytoconstituent responsible for the observed antioxidant activity and their mode of action.

Acknowledgements:

The Authors are thankful to the management of VISTA, Chennai and GIET School of Pharmacy, Rajahmundry for offering their kind support to carry out this study.

Conflict of interests:

The authors declared no conflict of interest.

References